Fabrication and characterization of a recombinant fibronectin/cadherin bio-inspired ceramic surface and its influence on adhesion and ossification in vitro

Abstract

This study has investigated the effects of a bio-inspired ceramic surface modified with a novel recombinant protein on surface parameters and cell behavior. The surface of a biphasic calcium phosphate ceramic was functionalized with a recombinant protein spanning the fragments of fibronectin module III7-10 and extracellular domains 1 and 2 of cadherin 11 (rFN/CDH) using a dimethyl-3,3'-dithiobispropionimidate cross-linking method. The surface was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and protein adsorption and surface density measurements. The material exhibited desirable properties for cell adhesion and proliferation. The effects of the surface on the adhesion and proliferation of human mesenchymal stem cells (hMSC) were investigated using a cell adhesion centrifugal assay and the 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The data demonstrated that the adhesive capacity and proliferation rate were significantly improved as compared with fibronectin and cadherin positive controls. Moreover, the rFN/CDH bio-inspired ceramic surface also induced osteoblastic differentiation, as evidenced by the higher alkaline phosphatase activity and osteocalcin mRNA expression level of hMSC cultured in osteogenic media for 7-10days. Furthermore, a functional blocking assay with a site-specific antibody against phosphotyrosine 397 (pY397) of focal adhesion kinase revealed that pY397 is involved in adhesion and ossification. These results suggest that the rFN/CDH bio-inspired BCP surface possesses enhanced functionality in adhesion, proliferation and ossification and may be a promising scaffold for tissue engineering.